Physiologically active polypeptides are useful as therapeutic agents for specific diseases. However, they are unstable when administered into blood, and a sufficient pharmacological effect can rarely be expected. For instance, physiologically active polypeptides having a molecular weight of less than 60,000 administered into blood are mostly excreted into urine by renal glomerular filtration, and their use as therapeutic agents is not expected to give a significant therapeutic effect and often requires repeated administration. Some other physiologically active polypeptides are degraded by hydrolases and the like existing in blood, thereby losing their physiological activities. Further, some exogenous physiologically active polypeptides have physiological activities effective for the treatment of diseases, but it is known that such exogenous physiologically active polypeptides and physiologically active polypeptides produced by recombinant DNA techniques sometimes induce immunoreaction when administered into blood to cause serious side-effects such as anaphylactic shock owing to the difference in structure between them and endogenous physiologically active polypeptides. In addition, some physiologically active polypeptides have physical properties unsuitable for use as therapeutic agents, e.g. poor solubility.
One of the known attempts to solve these problems in using physiologically active polypeptides as therapeutic agents is to chemically bind at least one molecule of an inactive polymer chain to physiologically active polypeptides. In many cases, desirable properties are conferred on the polypeptides or proteins by chemically binding polyalkylene glycols such as polyethylene glycol to them.
For example, superoxide dismutase (SOD) modified with polyethylene glycol has a remarkably prolonged half-life in blood and shows a durable action [Pharm. Res. Commun., Vol. 19, p. 287 (1987)]. There is also a report of modification of granulocyte colony-stimulating factor (G-CSF) with polyethylene glycol [J. Biochem., Vol. 115, p. 814 (1994)]. Gillian E. Francis, et al. summarized examples of polyethylene glycol-modified polypeptides such as asparaginase, glutaminase, adenosine deaminase and uricase [Pharm. Biotechnol., Vol. 3, Stability of Protein Pharmaceuticals, Part B, p. 235 (1992), Plenum Press, New York]. Further, it is known that modification of physiologically active polypeptides with polyalkylene glycols give effects such as enhancement of thermal stability [Seibutsubutsuri (Biophysics), Vol. 38, p. 208 (1998)] and solubilization in organic solvents [Biochem. Biophys. Res. Commun.: BBRC, Vol. 122, p. 845 (1984)].
With regard to the methods for binding polyalkylene glycols to peptides or proteins, it is known to introduce an active ester of carboxylic acid, a maleimido group, a carbonate, cyanuric chloride, a formyl group, an oxiranyl group or the like to an end of a polyalkylene glycol and bind it to an amino group or a thiol group in a polypeptide [Bioconjugate Chem., Vol. 6, p. 150 (1995)]. These techniques include the binding of a polyethylene glycol to a specific amino acid residue in a physiologically active polypeptide, which causes enhancement of stability in blood without impairing the biological activities of the peptide or protein. Examples of the polyethylene glycol modification specific to amino acid residues in physiologically active polypeptides include the binding of a polyethylene glycol to the carboxyl terminus of a growth hormone-releasing factor through norleucine as a spacer [J. Peptide Res., Vol. 49, p. 527 (1997)] and the specific binding of a polyethylene glycol to cysteine introduced to the 3-position of interleukin-2 by recombinant DNA techniques [BIO/TECHNOLOGY, Vol. 8, p. 343 (1990)].
Many of the above polyalkylene glycol-modified polypeptides are obtained by binding of linear polyalkylene glycols. However, it has been found that binding of branched polyalkylene glycols is preferable for obtaining chemically modified polypeptides having a high activity. It is generally known that the durability of a chemically modified polypeptide in blood is increased as the molecular weight of a polyalkylene glycol is higher or the modification ratio higher [J. Biol. Chem., Vol. 263, p. 15064 (1988)], but in some cases, the physiological activity of a physiologically active polypeptide is impaired by raising the modification ratio. This is partly because a specific amino group or thiol group in the physiologically active polypeptide which is necessary for its physiological activity is modified with a chemical modifier. For example, it is known that the physiological activity of interleukin-15 lowers according to the modification ratio [J. Biol. Chem., Vol. 272, p. 2312 (1997)].
On the other hand, it is difficult to synthesize high molecular weight polyalkylene glycols having a uniform molecular weight distribution and a high purity. In the case of monomethoxypolyethylene glycols, for example, contamination with diol components as impurities is known. Accordingly, attempts have been made to prepare high molecular weight modifiers by branching currently available polyalkylene glycols having a narrow molecular weight distribution and a high purity. Such attempts provide chemically modified polypeptides having a high physiological activity with a high durability retained even with a decreased modification ratio. Further, it is considered that a larger part of the surface of molecules of physiologically active polypeptides can be covered with polyalkylene glycols by branching of the polyalkylene glycols. For example, double-chain polyethylene glycol derivatives prepared by using cyanuric chloride as the group having a branched structure are known (Japanese Published Unexamined Patent Applications Nos. 72469/91 and 95200/91). In this case, a methoxypolyethylene glycol having an average molecular weight of 5,000 is utilized, but this compound has the problem of toxicity due to the triazine ring. Japanese Published Unexamined Patent Application No. 153088/89 discloses that a chemically modified polypeptide having a high activity can be obtained from a comb-shaped polyethylene glycol which is a copolymer of polyethylene glycol and maleic anhydride at a lower modification ratio compared with a linear polyethylene glycol. However, this compound has many reactive sites for a polypeptide, which causes impairment of the physiological activity of a physiologically active polypeptide, and has an ununiform molecular weight distribution. Also known are a compound having two polyethylene glycol chains through a benzene ring prepared by using cinnamic acid as a material (Japanese Published Unexamined Patent Application No. 88822/91) and compounds having two polyethylene glycol chains prepared by using lysine as a material (WO96/21469, U.S. Pat. No. 5,643,575).
As illustrated by the above examples, compounds having two polyalkylene glycol chains are known, but those having three or more polyalkylene glycol chains have not been produced. Although U.S. Pat. No. 5,643,575 suggests a three-branched, water-soluble, non-antigenic polymer, it contains no disclosure of the method for producing the three-branched compound or of specific examples and provides no information about the excellent effect of the three-branched compound.
There exists a need for a chemically modified polypeptide with improved durability which retains the activity of the physiologically active polypeptide and whose renal glomerular filtration is suppressed. In order to produce the chemically modified polypeptide exhibiting such properties, there is also a need for a modifier with a low toxicity and an improved stability which has an excellent molecular size-increasing effect and a narrow and uniform molecular weight distribution.